Molecular Markers for Biomass Traits: Association, Interaction and Genetic Divergence in Silkworm Bombyx mori

Improvement of high yielding, disease resistant silkworm strains became imminent to increase production of silk, which is a major revenue earner for sericulturists. Since environment interacts with phenotype, conventional breeding did not result in commendable yield improvement in synthetic strains of silkworm, Bombyx mori. Identification of DNA markers associated with different economically important biomass traits and its introgression could assist molecular breeding and expression of stabilized high yielding characters, but genetic basis of most quantitative traits in silkworm is poorly understood due to its polygenic control. Correlation analysis (R = 0.9) revealed significant interrelation among biomass traits viz., larval duration (TLD), larval weight (LWT), cocoon weight (CWT), shell weight (SWT), shell ratio (SR) and floss content. PCR using inter simple sequence repeat (ISSR) primers revealed 92% polymorphism among 14 tropical and temperate strains of B. mori, with average diversity index of 0.747. Stepwise multiple regression analysis (MRA) selected 35 ISSR markers positively or negatively correlated with different biomass traits, illustrated polygenic control. ISSR marker 830.81050bp was significantly associated with LWT, CWT, SWT, SR and floss content, indicated its pleiotropic role. Two ISSR markers, 835.51950bp and 825.9710bp showed significant association with floss content and TLD. These markers were segregated in F2 generation and Chi-square test confirmed (χ2 = ~45; P < 0.05) its genetic contribution to the associated biomass traits. Strains, with both positively and negatively correlated markers, had intermediate mean value for biomass traits (eg. SWT = 0.17 ± 0.014 g in GNM and Moria) indicated interaction of loci in natural populations. Low yielding Indian strains grouped together by Hierarchical clustering. Chinese and Japanese strains were distributed in the periphery of ALSCAL matrix indicated convergence of genetic characters in Indian strains. Average genetic distance between Chinese strains and Indian strains (0.193) significantly (P < 0.01) varied from that between Chinese and Japanese strains. Interaction of loci and allelic substitutions induced phenotypic plasticity in temperate B. mori populations on tropic adaptation in India. These outcomes show possibility to combine favorable alleles at different QTL to increase larval, cocoon and shell weight.


Introduction
Sericulture or cultivation of silkworms is an agro industry, producing commercially valuable silk on which scores of farmers of tropical and temperate Asian countries rely for their revenue. Domesticated silkworm Bombyx mori (Insecta: Lepidoptera: Bombycidae) is monophagous and feeds exclusively on leaf of mulberry, a hardy plant belongs to the genus Morus (Family: Moraceae). Silkworm germplasm encompass around 3000 genotypes having its origin in temperate and tropical countries (Nagaraju et al. 2000). B. mori gene pool is broadly categorized in to low yielding and high yielding strains. Low yielding strains are adapted to tropical conditions and are non-diapausing while high yielding strains adapted to temperate climate and undergo embryonic diapause. High yielding strains have higher cocoon weight, cocoon-shell weight, shell ratio and better yarn qualities in comparison to low yielding strains (www.silkgermplasm.com) but are highly susceptible to diseases. India, being a tropical country utilizes low yielding native strains, and breeds developed from Japanese and Chinese strains of B. mori for silk production. Silkworm breeding strategy is aimed at developing vigorous breeds and hybrids to meet twin demand of high survival and high production of quality silk. Though phenotypic characters depict variation, interaction of environment modifi es its expression. Therefore, gene tagged breeding would be a promising approach to combine best quality of temperate high yielding strains with tropical disease resistant ones. Since most quantitative economic characters are controlled by interactions between multiple genes and environment, identifi cation of gene(s) associated with a specifi c character is an enigma. Genetic analysis of quantitative traits became possible due to availability of large number of molecular markers to which QTL is associated. Genetic characterization of various silkworm strains of diversifi ed phenotype and identifi cation of gene markers for each of the economically important characters could contribute to develop strategy for future breeding programs using marker-assisted selection. Genes that contribute to naturally occurring variations in quantitative traits of B. mori strains may not vary in the mapping population too. Hence, association of markers with different traits and its distribution in natural populations are to be detected. Molecular marker systems like Random Amplifi ed Polymorphic DNA (RAPD; Nagaraja and Nagaraju, 1995;Chatterjee and Pradeep, 2003), Restriction Fragment Length Polymorphism (RFLP; Sethuraman et al. 2002), microsatellites (simple sequence repeats; SSR; Prasad et al. 2005) and inter SSR (ISSR; Reddy et al. 1999; highlighted the utility of molecular markers in silkworm fi ngerprinting and analysis of markertrait association. These potential marker systems and single nucleotide polymorphism (SNP) markers were also used to generate molecular maps of B. mori (Goldsmith et al. 2005;Nagaraja et al. 2005;Yamamoto et al. 2006).
ISSR products resolved on agarose gel are dominant markers and the system offers rapid production of a large number of markers in costeffective manner. ISSRs are DNA fragments located between adjacent, oppositely oriented microsatellites amplifi ed by PCR using microsatellite core sequences and a few selective nucleotides as a single primer. As short repeats are ubiquitously distributed in eukaryotic genome, single primers of di-, tri-, tetra-and penta nucleotide simple sequence repeats are employed for amplifi cation of markers. ISSR markers, evolve faster as they are genomic regions with microsatellites that exhibit variable mutation rates and high level of polymorphism (Schlotterer, 2000), due to DNA polymerase slippage or DNA double strand breakage (Strand et al. 1993;Jankowski et al. 2000). After initial identification of ISSRs in humans (Zietkiewicz et al. 1994), its greater usefulness in fingerprinting has been established in different organisms including plants (Nagaoka and Ogibara, 1997;Agaki et al. 1996;Deshpande et al. 2001) and insects (Ehtesham et al. 1995;Reddy et al. 1999;Abbot, 2001;Chatterjee et al. 2004;Vijayan et al. 2006;Pradeep et al. unpublished). ISSR markers are usually located in non-coding regions and are selectively neutral. Because ISSR primers generate multi locus fi ngerprinting profi le, ISSR analysis has been applied in studies involving genetic identity, parentage, clone and strain identifi cation as well as gene mapping studies (Vogel and Scolnik, 1997). Considering these advantages of ISSR primers, this marker system was used to identify molecular markers associated (not linked) with biomass traits and to analyze genetic variability among few strains of B. mori.
Association of molecular markers with different economic traits or disease resistance was studied mostly in crops such as chick pea (Ratnaparkhe et al. 1998), rice (Hussain et al. 2000) and maize (Domeniuk et al. 2002). Molecular markers for antibody response in chickens (Yonash et al. 2000) and gene for larval growth in Drosophila (Becker et al. 2001) were also reported. Due to the economic importance of silkworms and need for high yielding disease resistant strains, conventional breeding techniques has to be supported by directional selection utilizing yield associated molecular markers. Hence investigations on association of various molecular markers with different yield attributes had initiated in silkworms (Sethuraman et al. 2002;Chatterjee and Mohandas, 2003;Chatterjee and Pradeep, 2003;Gaviria et al. 2006) (Table 1). Since each quantitative trait is under regulation of different genes, their associations with different traits have yet to be established. Association of molecular markers with different traits was studied in different organisms using methods such as MRA, bulk-segregant analysis (BSA) and discriminant function analysis (DFA). While MRA provided statistical association of markers, based on its correlation with traits (Virk et al. 1996;Yonash et al. 2000), BSA could identify markers for a specifi c trait from segregating population (Michaelmore et al. 1991). On the other hand, DFA used a group co-variance matrix, adopting stepwise selection of independent variables. DFA facilitated identifi cation of a molecular marker that revealed geographical isolation of Japanese strains of B. mori from mainland (Sino-Russian-Indian) populations (Chatterjee and Pradeep, 2003). In the

Genetic material and DNA extraction
Fourteen strains of B. mori, originating from India, China and Japan used in this study, were maintained at Central Sericultural Germplasm Resource Centre, Hosur, Tamil Nadu, India where strains were reared for more than 10 years at standard rearing conditions of 25 ± 2 o C temperature and 75 ± 3% relative humidity. Phenotypic data collected from three replications of the rearing (n = 30 each) is given in Table 2. To study inheritance of markers, two crosses were made between low yielding strains Pure Mysore/C'nichi females and high yielding strain NB1 (male). F 1 generations were raised and moths were allowed for sister-brother crossing (self mating) to develop F 2 generation of both the crosses. Phenotypic data of each F 2 individual was collected (n = 32). Genomic DNA of different B. mori strains (n = 30 individuals each) and F 2 generation individuals was extracted from moths by phenol:chloroform: isoamyl alcohol method (Suzuki et al. 1972). DNA was dissolved in TE (Tris-EDTA) buffer (pH 8.0) and diluted and quantifi ed to a concentration of 10 ng per micro Liter against standard uncut lambda DNA (10 ng/micro Liter).

Statistical Analyses
Data generated by ISSR primers were used for analysis using the program SPSS v 11.5 (M. J. Norusis, SPSS Inc., Chicago). Banding pattern generated by each primer was scored into a matrix  with presence of amplifi cation product as "1" and absence as "0" and this binary matrix was used for analysis. Biomass traits considered for this study were total larval duration from hatching to initiation of spinning (TLD), maximum weight attained by fi nal instar larva (LWT), cocoon weight (CWT), cocoon shell weight (SWT), shell ratio (SR % = SWT/CWT × 100), outer loose layer of silk over the cocoon or fl oss (%) and reeling silk waste (%). Differences between mean estimates of traits among 14 strains were assessed by ANOVA. Interrelation between different traits was assessed by correlation analysis. Multiple regression analysis (MRA) was used for identifi cation of markers associated with different biomass traits with molecular markers as independent variable and biomass trait estimates as dependent variables. Stepwise variable entry and removal used in MRA examined the variables at each step for entry or removal. MRA used the model for regression equation with F values of 0.045 and 0.099 as limiting frame for stepwise selection and rejection of the independent variable (Affi fi and Clark, 1984). Beta statistics was calculated for each marker and is defi ned as standardized regression coeffi cient = BSx/Sy, where B is regression coeffi cient, Sx and Sy are the standard deviations of independent (x) and dependent (y) variables (Affi fi and Clark, 1984). Student's t-test was performed to test signifi cance between mean trait estimates of strains where specifi c markers were present and absent.
Single factor ANOVA (SFA) was performed to establish association of markers with biomass traits of different strains as well as of F 2 generation individuals. The procedure produces a one-way analysis of variance for a quantitative dependent variable (trait) by a single factor (independent) variable (molecular marker). Single marker analysis (SMA) was performed with MRA selected markers as the classifying variable to identify QTLs associated with biomass traits in F 2 generation. Chi-square (χ 2 ) test was performed to examine goodness-of-fi t between marker-locus contributions in F 2 generation. Effect of interaction of MRA selected markers on its association with different traits was assessed by analyzing level of significance (Students' t-test) in difference between estimates of each trait.
In order to analyze genetic divergence data developed from dominant ISSR markers, genetic similarity coeffi cients among 14 strains were estimated from the binary data by Heirarchical cluster analysis using Jaccard measure, Dice measure and Sokal and Sneath measure. Jaccards' coeffi cient was GD J = 1-[N 11 /(N 11 +N 10 +N 01 )], Dice coeffi cient was GD D =1-[2N 11 /(2N 11 +N 10 +N 01 )] where N 11 is the number of bands present in both individuals, N 00 is number of bands absent in both the individuals, N 10 and N 01 are number of bands present only in the individual and N represents the total number of bands. Sneath and Sokal, (1973) coeffi cient for genetic distance between genotypes i and j (Dij) was determined by Dij = 1-Sij = 1-[a + d/(a + b + c + d)] where Sij = similarity coeffi cient; a = number of matches 1,1; b = number of matches 1,0; c = number of matches 0,1 and d = number of matches 0,0. Genetic distance was calculated as (1-Similarity coeffi cient). Dendrograms were resolved from similarity matrices to compare genetic distance among strains based on different algorithms. In order to analyze distribution of silkworm genotypes from India, China and Japan, multidimensional scaling of ISSR data from 14 strains was done using ALSCAL program. In this method, a dissimilarity matrix was created using Euclidean distance and was used for stimulus configurations of the data using the classical Young-Householder multidimensional scaling procedure (Young et al. 1984;Young and Harris, 1990).

Results
Mean estimates of biomass traits, country of origin and diapause behavior of 14 different strains of B. mori is given in Table 2. Among the strains, signifi cant (ANOVA; P < 0.005) variation was observed within estimates of biomass traits such as LWT, CWT, SWT, SR, fl oss as well as silk waste.

Interrelation between biomass traits
Correlation analysis showed positive correlation (R = 0.916) among mean estimates of LWT, CWT, SWT and SR (Table 3). SWT (R = 0.923) and SR (R = 0.742) showed significant increase with increase in CWT. Increase in SWT (R = 0.554) and SR (R = 0.607) showed highly significant (P < 0.001) increase with TLD but this relation was not apparent with other parameters. Floss content showed negative relation with increase in LWT and CWT (R = _ 0.786). Quantity of silk waste did  (Table 4). Based on binary matrix of ISSR profi le, step wise MRA identifi ed 35 ISSR markers associated with different biomass traits. Details of MRA and beta statistics with signifi cance are given in Table.5. In the fi rst step, MRA selected ISSR marker 830.8 1050bp for LWT, CWT, SWT and SR. On linear regression, this marker was negatively correlated with increase in estimates of these parameters (R 2 = ~0.8). Subsequently, MRA selected 851.1 1700bp for LWT (R 2 = 0.930), 810.2 1350bp for CWT (R 2 = 0.948) and 834.11 900bp for shell weight (R 2 = 0.925) and shell ratio (R 2 = 0.826). For TLD, 825.9 700bp was selected initially (R 2 = 0.738), followed by 835.11 1000bp (R 2 = 0.738). The marker, 825.9 700bp was present in low yielding strains except in Pure Mysore and absent in most of the high yielding strains. Highest number of markers (seven) was selected for fl oss content, of which four were selected with negative correlation and three with positive correlation. The marker, 830.8 1050bp was selected in the fi rst step for fl oss content also but found as positively correlated (R 2 = 0.631). For silk waste, 881.4 2000bp was selected in the fi rst step and it showed weak negative correlation (R 2 = 0.557). All together, 16 markers were positively correlated with increase in estimates and 19 markers were negatively correlated (Table.5).

Association of Markers with Traits
Student's t-test confi rmed signifi cance (P < 0.005) in variation between phenotype estimates of strains showing presence and absence of marker associated with each trait (Table. 6). The marker 830.8 1050bp showed highly signifi cant (P < 0.0003) association with low estimates of LWT, CWT, SWT, SR and with high estimate of fl oss content (P < 0.005). This marker was present in low  Table 2. M-molecular marker (Massruler, Fermentas). yielding strains of B. mori (Nistari, C'nichi, Pure Mysore, GNM and Moria) and was conspicuously absent in high yielding strains (Fig. 1). Of the other markers, 835.9 1050bp , 836.4 2300bp and 835.5 1950bp showed signifi cant (P < 0.02 to 0.002) positive association with high estimates of TLD, LWT and floss content respectively. All other markers showed signifi cant negative relation with different traits (Table 6). In F 2 , MRA selected markers segregated in 1:1 ratio, except 835.5 1950bp , in 3:1 ratio. Single factor ANOVA showed highly significant (P < 0.000) association of 830.8 1050bp with LWT, TLD, CWT, SWT, SR and fl oss content of different strains and in F 2 population (Tables 7A  & B Effect of interaction of two markers associated with a specific character was analyzed by comparing estimates of traits of different strains using t-test. Mean values of TLD, CWT, SWT, silk waste and fl oss content of strains either with a negatively correlated marker, or with a positively correlated marker and strains with both the negative and positive markers are given in Table 9. B. mori strain GNM with both 830.8 1050bp and 810.2 1350bp had intermediate CWT (1.19g), which showed signifi cant (P < 0.007) variation from CWT of strains with 830.8 1050bp alone but did not differ signifi cantly from those with 810.2 1350bp . In shell weight and silk waste, intermediate values showed signifi cant (P < 0.005) variation from high and low estimates whereas in fl oss content, intermediate value of C'nichi signifi cantly (P < 0.06) varied from low fl oss content strains but did not vary from high fl oss content strains (P < 0.164). LWT of strains having marker 830.8 1050bp was 2.265 ± 0.378g whereas that of strains with 836.4 2300bp was 3.987 ± 0.153g. No strains used had both the markers (830.8 1050bp and 836.4 2300bp ) together. F 2 individuals (of Pure Mysore x NB1 cross) in which both these markers were present had LWT of 2.914 ± 0.424 g (equivalent to mid-parent value), which was signifi cantly (P < 0.0001; Student's t-test) higher than LWT (2.314 ± 0.359 g) of individuals without these markers. TLD of the strain HU204, which had both 825.9 710bp and 835.11 1050bp , was higher (631 hours) than other groups.

Genetic Divergence Between Low Yielding and High Yielding Strains
Polymorphic profi le generated by ISSR primers from 14 different strains of B. mori (Table 4) was further analyzed by Hierarchical clustering. Three different algorithms viz., Jaccard measure, Dice measure and Sokal and Sneath measure were used to evaluate genetic relations among the 14 strains. Jaccard and Dice measures clustered strains in similar pattern but were different from grouping by Sokal and Sneath measure ( Figs. 2A and B). Three Indian low yielding strains Nistari, Pure Mysore and Moria were grouped

A B
Most of the Japanese and Chinese strains were distributed in periphery of matrix (Fig. 3).

Discussion
Biomass traits showed significant variability among different strains of silkworm, B. mori. Correlation matrix showed high coeffi cient value (>0.9) between larval weight and cocoon/ shell weight indicated contribution of larval weight to formation of cocoon (pupa and its shell). Total larval duration contributed signifi cantly to increase in cocoon and shell weight. In silkworms, larva is the only feeding stage in the life cycle and it accumulates energy for all life stages and contributes to formation of cocoon, pupa and moth as well as reproductive processes. In insects, critical larval weight together with larval duration accomplishes endocrine-mediated metamorphic processes (Pradeep et al. 2000;Truman 2005) but the process is under genetic control (Dubrovskaya et al. 2004). Correlation between quantitative traits and biochemical parameters had reported earlier in B. mori (Shibukawa et al. 1986;Chatterjee et al. 1993). Signifi cant correlation among biomass traits refl ects interrelation among physiologically important processes. Hormones coordinate multiple developmental and physiological processes and are major determinants underlying phenotypic integration (Flatt et al. 2005). High shell weight is accompanied by low fl oss content indicated that silk formed by the larvae is utilized to its maximum for shell formation in high yielding strains. Silk waste is determined after reeling of cocoons, which include mechanical processing that causes more wastage of fi lament. This may be the reason for lack of correlation of silk waste with biomass traits. ISSR primers showed large diversity index (DI) of 0.747, of which dinuleotide repeats revealed higher level of diversity among the strains. This is consistent with presence of large number of dinucleotide repeats in B. mori genome (Prasad et al. 2005). Variability in number of markers generated by different primer systems was reported earlier in plant systems as well (Akagi et al. 1996). Association of different ISSR markers with biomass traits was established using MRA. Regression analysis was used to associate molecular markers with economic traits in agricultural crops (Barbosa-Neto et al. 1996;Lynch, 1999;Yonash et al. 2000;He et al. 2002) and for different quantitative traits in B. mori (Table 1). In B. mori, 45 dominant markers (ISSR- Chatterjee and Mohandas 2003;RAPD-Chatterjee and Pradeep, 2003) and 32 co-dominant markers (RFLP -Sethuraman et al. 2002;STS-Mohandas et al. 2004;AFLP -Gaviria et al. 2006) were found associated with CWT and 26 dominant markers and 15 RFLP markers, with total larval duration. An ISSR marker associated with long larval duration was identifi ed from an inbred population of B. mori after artifi cial selection . Stepwise MRA selected the markers based on its contribution to trait and consequently, interaction and additive effect of multiple markers on a specific trait could be   assessed. MRA identifi ed 35 ISSR markers in association with different biomass traits. These markers were correlated negatively or positively with estimates of phenotypic characters. Test of significance on association of markers with different traits reduced number of significant markers to 12. In the fi rst step of MRA, the marker 830.8 1050bp was selected for larval weight, cocoon weight, shell weight and fl oss content. This marker was exclusively present in strains with low estimates of biomass traits and high fl oss content. Negative association of this marker with LWT and cocoon characters and its positive association with fl oss content corroborate with negative correlation (R = -0.754) of fl oss content with LWT/cocoon/ shell weight. Such pleiotropic associations of molecular markers with different cocoon characters and yield attributes were illustrated in B. mori (for references see Table 1). Identifi cation of several markers for each trait assigns interactive effect of selected independent variables on the dependent variable (Cochran, 1938;Steel and Torrie, 1980), which in turn substantiates multigenic control of the biomass traits (Shibukawa et al. 1986). Single factor ANOVA showed signifi cant association of 830.8 1050bp with biomass traits among different strains. In F 2 generation, the marker 830.8 1050bp was segregated at 1:1 ratio. Notedly, in silkworm B. mori, recombination occurs only in the homogametic males and is absent in the heterogametic  females. Any F 2 individual can not be homozygous for both maternal and paternal dominant markers on the same autosome (Nagaraju and Goldsmith, 2002). As the ISSR marker on agarose gel is dominant, it could not distinguish a heterozygote. In F 2 , low yielding homozygote individuals with 830.8 1050bp marker and high yielding individuals without this marker appeared in equal proportion (1:1). Though major loci for biomass traits are sex-linked is to be analyzed, recent observations indicated distribution of markers thoroughout the Z chromosome and few markers in the W chromosome (Nagaraja et al. 2005). On the other hand, the marker 835.5 1950bp for fl oss content appeared at true Mendelian ratio of 3:1 ratio which indicates dominant nature of this locus and the high fl oss content in tropical strains. Chi-square values (χ 2 = 44; P < 0.05) revealed signifi cant genetic contribution of the marker 830.8 1050bp to LWT, CWT and SR. Association of 830.8 1050bp with different biomass traits refl ects pleiotropic effect of the locus on various traits with large effect on LWT that showed positive correlation with cocoon weight and shell weight. Correspondingly, signifi cant genetic association of 836.4 2300bp with LWT was also noticed. Though SMA showed signifi cant association of 825.9 710bp with TLD, 830.8 1050bp and 835.5 1950bp with fl oss content and 830.8 1050bp with SWT, Chi-square values were insignifi cant. This indicates that association of these markers is infl uenced by causes other than genetic factors. A closest marker fl anking a QTL may not be tightly linked to a gene (Michelmore, 1995), which may be due to recombination between the marker and QTL (Collard et al. 2005). Further, shell and fl oss are made of silk proteins, for which amino acid budgeting is made from amino acid pool present in the larval haemolymph. Depending on nutrient quality of mulberry leaf and environmental factors, availability of amino acids in larval haemolymph varies, which signifi cantly affects silk production (Sehnal and Akai, 1990) and thereby infl uences shell weight and fl oss content.
Biomass traits showed a switch in phenotypic expression according to presence or absence of markers associated with a specific trait. For instance, shell weight ( (Table 9). Of the two markers selected for each trait in this study, one of them is negatively correlated and the other is positively correlated. Combined effect of these markers on phenotypes appeared as intermediate as these loci affect characters in opposite directions (Falconer and Mackay, 1996). Such markers are signifi cant as they contributed genetically in opposite directions in two different (temperate and tropic) environments. In F 2 generation, LWT varied signifi cantly (P < 0.0001) between individuals with  830.8 1050bp and 836.4 2300bp and those without these markers. These loci had opposite effects on LWT in the parents, Pure Mysore and NB1. Since the QTL and the markers inherited together in F 2 progeny, mean of the group with the markers signifi cantly varied (P < 0.001) from that of the group without the marker. This indicates that the marker loci 830.8 1050bp and 836.4 2300bp are associated to a QTL controlling LWT, though linkage has to be established. Signifi cant interactions between QTLs were noticed in soybean in which height variation at one locus is conditional upon another specifi c allele (Lark et al. 1995) whereas ovariole number in Drosophila species is under control of sign epitasis of QTLs (Orgogozo et al. 2005).
Interaction of QTLs was also reported for different traits in various organisms including number of abdominal bristles and sex comb teeth in Drosophila (Long et al. 1995;Tatsuta and Takano-Shimizu, 2006), fruit traits of tomato (Paterson et al. 1991), seed weight in cowpea and mung bean (Fatokun et al. 1992), maize infl orescence traits (Doebley and Stec, 1993), and protein content in soybean (Tajuddin et al. 2003). Notably, intermediate trait values and presence of both negatively and positively correlated markers are characteristics of B. mori strains originated in temperate regions of Asia (China and Japan). These strains were either brought to tropical conditions of India or evolved from Japanese/ Chinese parents for commercialization. Localized multiplication over a long period might have resulted in allele substitutions, which are common in tropical strains of B. mori (Hirobe, 1968;Gamo, 1983). Allele substitutions lead to phenotypic plasticity (Ungerer et al. 2003) as an adaptation in the tropics. In total gene pool of B. mori comprising several strains, genetic markers interacted to control expression level of fi tness traits according to the needs during adaptation. More over, impact of a locus associated with a specifi c character could be augmented or weakened by presence of another associated locus. Intermediate phenotype and genetic setup of temperate strains under tropical conditions refl ect genetic differentiation of new silkworm populations. By defi ciency mapping of QTL affecting longevity in natural population of Drosophila, Pasyukova et al. (2000) suggested that QTL contributing to variation in a quantitative trait between two particular strains contribute to variation of the trait in nature, to which present observations on interactions of ISSR loci and its associa-tion with biomass traits in B. mori strains corroborate. Larval duration is an exception, which was significantly higher in Hu204, in which both 825.9 710bp and 835.11 1050bp were present. This may be due to small genetic effect of individual QTLs, which are sensitive to the environment (Mackay, 2004). In B. mori, larval duration is infl uenced by loci sensitive to selection ) and alleles of juvenile hormone responsive gene , but the intensity of interaction of environment with them is not known. More over, single QTLs could be fractionated into multiple linked QTLs as found in Drosophila, effects of which could not be equal on a trait (Pasyukova et al. 2000;Harbison et al. 2004). In insects, larval duration is infl uenced not only by genetic factors but humoral and environmental cues also (Sehnal, 1985). Impact of marker x environment interaction to determine total larval duration in B. mori is to be analyzed in detail.

Genetic Divergence
Genetic markers represent genetic differences between strains and reveal sites of variation in DNA (Winter and Kahl, 1995;Jones et al. 1997). Several dominant ISSR markers resolved on agarose gel were used for genetic divergence analysis. Earlier studies revealed relative advantages of different algorithms based on grouping of maize inbreds using RFLP data (Ajmone-Marson et al. 1992;Mumm et al. 1994). Silkworm strains used in this study are of Asian origin. It is well known that most of these strains were descent from China in the long past and adapted to diverse climates, point to genetic closeness among them. This indicated a necessity of more than one algorithm to examine genetic divergence within these closely related silkworm populations. Hierarchical cluster analysis grouped low yielding Indian strains and high yielding temperate strains independently. Nistari is an original tropical strain of Indian origin and its rearing has been practiced in Ganges river valley since more than a century (Mukherjee, 1912). Though Pure Mysore is a tropical, low yielding Indian strain, its origin is not clear. Low genetic distance and clustering of Pure Mysore with Nistari refl ect that these strains are genetically closer. Long association with tropical conditions and stabilization through continuous commercialization made Pure Mysore a segregant population of India. Though Pure Mysore is adapted for tropical climate, larval duration is longer as in temperate strains of B. mori. The marker 825.9 710bp selected for TLD was present in all the low yielders but absent in Pure Mysore, and other strains of temperate origin. This is consistent with our earlier observation on presence of TLD associated RAPD marker UBC89.5 1500bp in Pure Mysore and long duration high yielding strains of temperate origin and its absence in tropical low yielding strains (Chatterjee and Pradeep, 2003). Molecular data on the rare RAPD locus and ISSR locus (present observations) associated with larval duration and alleles associated with juvenile hormone responsive genes  supported the presumption that Pure Mysore is a hybrid of Chinese and Japanese strains of temperate origin (Datta, 1984). C'Nichi is originally a diapausing strain of Japan but adapted to Indian conditions and became a non-diapausing strain. Isolation of C'nichi in the dendrograms signifi ed its stabilization as an independent strain after long-term adaptation to tropic climate. High yielding strains, which grouped together, were originated from Japanese or Chinese parental strains and have been used for sericultural activities in India since 1960s. Average genetic distance between Chinese and Indian strains varied signifi cantly (under Sneath and Sokal measure) when compared with that of Chinese-Japanese and Japanese-Indian strains. This indicates segregation and genetic differentiation of those Chinese strains under tropical conditions of India by continuous localized multiplication. This was supported by the observation on ALSCAL matrix that indicated global distribution of genetic characters of Chinese and Japanese silkworm strains and its convergence in India.
Though marker-trait association studies have to be supplemented with linkage analysis, identifi cation of several potential markers that contribute to develop genetic characteristics of silkworm population and reveal genetic divergence within low and high yielding strains, could have potential practical utility in prospective silkworm breeding program.